Ethanol alters the function of several ligand-related ion channels including the 5-HT receptor. Potentiation of 5-HT, receptor function at intoxicating ethanol concentrations is thought to contribute to acute ethanol intoxication and alcohol drinking behavior. The receptor can be purified to homogeneity and this will allow us to address several important questions concerning the effect of ethanol on receptor structure and structural dynamics. Using purified and reconstituted 5-HT, receptor protein with selected point mutations we will perform specific experiments designed to test the hypothesis that ethanol enhances movement of the alpha-helical second transmembrane domain lining the channel pore of the 5-HT3 receptor to prolong total channel open time in the presence of low agonist concentrations. This hypothesis will be tested by site-directed spin-labeling (SDSL) and EPR measurement of: a) the secondary structure of the transmembrane II (TMII) region of the protein in the absence and presence of ethanol; b) ethanol-induced changes in mobilit of the side chains of residues within the TMII domain; c) accessibility of paramacnetic agents to TMII residues; and d) ethanol-induced changes in measured distance between site-directed spin labeled amino acids in the TMII region. These experiments will provide novel information about the structural basis of ethanol effects on ligand-gated ion channels. These studies will also provide the basis for future analysis of the relationship between receptor structure and ethanol sensitivity. Ultimately, we hope that this research will contribute to the development of pharmacological approaches to the treatment of alcohol abuse and alcoholism.